Final Reports (1999)

ES-Derived Neurons and Glia: a Novel Cell Line Model for CNS Testing

David I. Gottlieb, PhDWashington University School of Medicine, St. Louis, Missouri

Introduction

Mouse ES cells have great potential to reduce the use of animals in basic research and in the pharmaceutical industry. ES cells are capable of differentiating in vitro into a variety of cell types including neural, hematopoietic, vascular endothelial and cardiac cells. My laboratory has discovered a method for efficiently inducing ES cells to differentiate into neural cells (Bain et al, 1995); similar protocols have been developed in other laboratories (Fraichard et al, 1995; Strubing et al, 1995). Using our protocol we produce cultures consisting of neurons and glial cells. They are fully functional, forming, axons dendrites and functional chemical synapses between them. (Finley et al, 1996) The neurons utilize the neurotransmitters glutamic acid, GABA or glycine. In every way, these neurons resemble those found in dispersed cell cultures derived directly from the brain. It is therefore reasonable to explore whether these cultures could be used to evaluate compounds for toxicity.

Lead and alcohol are 2 toxins whose effects on neurons have been studied (Kern et al, 1993; Tiffany-Castiglioni, 1993; Heaton et al, 1994). We therefore analyzed the effect of these toxic substances on ES cell-derived neurons and glia.

Induction of differentiation - ES cells were subjected to an 8 day induction procedure which consisted of 4 days of culture as aggregates without RA followed by 4 days of culture in the presence of RA. To set up inductions, rapidly growing stock cultures of undifferentiated ES cells were trypsinized with 0.25% trypsin and 1 mM EDTA in saline to remove cells from the substratum. The detached cell suspension was triturated to yield a mixture of single cells and small clumps. To standardize cell input for the induction procedure, one quarter of the cells from a single confluent T25 flask were seeded per 10 ml of medium (standard medium described above) without LIF and beta-mercaptoethanol in a 100 mm diameter bacteriological (non-tissue culture) dish. Cell suspensions were cultured for 2 days during which time floating aggregates of cells formed.

The medium was changed and culture was continued for an additional 2 days. At this time, the medium was changed and, where indicated, supplemented with 5 x 10-7 M all-trans retinoic acid. After 2 additional days the medium was exchanged for fresh medium with RA for treated cultures but without RA for controls and culture continued for 2 more days. Aggregates which had been through the 8 day induction period were dissociated with trypsin (.25% with EDTA; 10 minutes) and plated on gelatin coated dishes.

Plating for toxicity experiments - Fully induced cells were plated into gelatinized 35 mm wells of a six-well dish. Approximately 1 x 106 cells per well were plated in 2 ml of the standard medium. Cells were cultured for 2 days without the addition of lead. Lead, at concentrations given in the text, was then added and culture continued for an additional 2 days.

Fixation and morphological evaluation - After 2 days incubation with lead the cells were fixed with 2% paraformaldehyde made up in PBS. After 1/2 hour fixation the fixative was carefully removed and the dishes washed with PBS. There is a systematic change in density of cells as a function of the distance from the center of the well. Therefore each well was marked with a ring at a specified distance from the center and counts performed within that ring. Fields were identified by moving the microscope stage equal distances without observing the field so as not to bias results. After choosing a field the neurons within it were counted.

Lead - Lead acetate (Fisher Scientific L33-250; lot # 976621) was used throughout these experiments; this was made up in aqueous solutions of 12.1 and 1.2 mM.

Ethanol - Pure ethanol was added to cultures in six wells to the indicated final concentration.

Results

In order to asses the effects of lead on neural cultures derived from ES cells, a series of cultures was set up in the 35 mm wells of 6 well cluster plates. Cultures in the wells were exposed either to no lead (controls) or to an increasing concentration of lead (10, 25, 50 and 100 micromolar). Duplicates of each concentration were run. In control cultures neurons and background cells are healthy and growing well. Neurons have extensive neurites which are firmly adhered to the background cells. The latter are well attached to the surface of the dishes and form a nearly confluent monolayer. Wells treated with 10 µM essentially look like the control wells. Cultures treated with 25 µM lead also show little difference relative to the control. Cultures treated with 50 µM show a noticeable difference. The most notable effect is on the background cells. Many of these are gone. There are still visible neurons although substantially fewer than the control. Wells exposed to 100 µM showed devastating effects from exposure to lead. Background cells were essentially wiped out. However, some neurons remained viable, had extended neurites, and remained attached to the bottom of the dish. Figures 1 and 2 are from 2 separate experiments and show the dramatic effects of 100 µM lead.

Figure 1

Effect of lead acetate on differentiated KD3 cells. Neurons were prepared in wells and incubated for 48 hours before the addition of lead. Cells were incubated for an additional 48 hours in the presence of lead, and then fixed.

One of the control wells, which did not receive any lead. Both the neurons and the background cells appear to be healthy and seem to be growing well.

Addition of 16 µL of lead acetate, resulting in a 100 µM concentration. Background cells are virtually wiped out, though few still remain. Some neurons nevertheless continue to appear healthy.

Figure 2

Effect of lead acetate on differentiated KD3 cells. Neurons were prepared in wells and incubated for 48 hours before the addition of lead. Cells were incubated for an additional 48 hours in the presence of lead, and then fixed.

One of the control wells. Both the neurons and the background cells appear to be healthy and seem to be growing well.

Addition of 100 µM lead acetate. Background cells are virtually wiped out, with only a few still remaining. Neurons nevertheless continue to be visible.

These qualitative observations were extended by making systematic counts of the neurons present after treating with 100 µM lead. Two experiments are shown (Table 1 and Table 2). The results in Table 1 show about a 4 fold decrease in the number of neurons present. The results in Table 2 also show a dramatic decrease. Here, the concentration of control cells was even higher than in Table 1 and was too dense to count. After lead treatment there was a dramatically lower density of neurons.

Table 1: Addition of Lead Acetate to ES Cell-Derived Neural Cultures

Control

100 µM

196

228

57

64

211

182

69

47

216

177

59

70

198

186

42

59

210

187

67

52

190

188

49

46

194

206

63

44

Avg = 202.1428

Avg = 193.4285

Avg = 58

Avg = 54.57142

S.D. = 10.00714

S.D. = 17.69987

S.D. = 9.712534

S.D. = 9.964221

AVG = 197.7857S.D. = 14.53472

AVG = 56.28571S.D. = 9.619120

Table 2: Addition of Lead Acetate to ES Cell-Derived Neural Cultures

Control

100 µM

Too dense to count

Too dense to count

93

104

85

97

92

92

103

102

105

104

100

109

104

115

Avg = 97.42857

Avg = 103.2857

S.D. = 7.546680

S.D. = 7.521398

AVG = 100.3571S.D. = 7.850596

Cultures treated with ethanol at concentrations 0.6%, 1.2% 1.8% and 2.4% showed variable results. We were unable to get repeatable results among wells with the same concentration. One cause of the variability is that ethanol was clearly being volatilized, making the concentration present in the wells uncertain. At highest concentrations ethanol was clearly having an effect on the neurons, but we were not able to quantify it reliably.

Conclusions

Our results show that lead has a dramatic effect on ES cell-derived neural cultures. At 100 µM lead, there was a severe loss of background cells. Studies from our lab (G. Bain and D.l. Gottlieb, unpublished observations) and another (Fraichard et al, 1995) indicate that many of these cells are glial. Numbers of neurons were dramatically reduced as well. These results show that cells derived from ES cells are sensitive to lead. Rat brain hippocampal cells are sensitive to lead in this concentration range (Kern, 1993). Thus ES cell derived neural cells have a lead sensitivity which resembles at least on well characterized type of CNS neuron. Interestingly, rat cortical cells are not sensitive to high concentrations of lead (Kern et al, 1993). Obviously there is great variability in neuronal sensitivity to lead.

Summary of the effectiveness of the research as relates to refining, reducing or replacing animals

ES cells replicate without limit in tissue culture. In principle an ES cell line derived from a single mouse embryo could provide suffficient cells for large-scale toxicological testing. The ability to efficiently differentiate ES cells into neurons and glia is now very well documented. The present results demonstrate that these cells are reasonable candidates for toxicological testing. With further testing and validation the system could replace many tests now being done either with animals or cells and tissues derived directly from them.